Experimental Study On Bu-Wang-San Affectingcognitional Function In Ovariectomized Rats

BackgroundAging is an important social and heathy problem in many countries incluing China.China is a big population country,the aging population is more serious.There are more than 10 percent sixty years or older population,so the number of dementia patients will be increasing significantly,especially in the older old.Alzheimer's disease is a neurodegenerative disorders correlated with age,approximately 60 percent in dementia.More and more olds are bothered by AD,and AD has been the fourth cause of old people's death next to cardiovascular diseases,cancer,stroke.An epidemiological investigation showed that female AD patients are obviously more than male patients with 1.5-3 times.It suggested that estrogen deficiency is associated with emergence and development of AD.Postmenopause women have a decrease in endogenous estrogen,which increases the incidence of AD.Estrogen replacement therapy(ERT)can ameliorate cognitional function.ERT provides a new method to prevent and treat AD.There are many aspects deserving to study,such as improving the function of cholinergic system,facilitating plasticity and transmission of synapse,anti-apoptosis of neuron,inflammation,et al.However,ERT may lead to unforeseen side-effects to subjects.This is the main cause to restrict the clinical application of ERT.Therefore,there are more and more attention to Chinese herbs with estrogenous effect.ObjectiveThe study used OVX as the model of memory impairment with estrogen deficiency.To study the mechanism of Bu-Wang-San to improve the cognitional function of postmenopausal women and provide theoretical foundation to clinical related disease.MethodsThe successful OVX rats were randomly divided into four groups.There are five groups in this study(n=12):SHAM control group,OVX group,OVX +E₂ control group(OVX+E₂),the high dosage BWS group(OVX+ BH),the low dosage BWS group(OVX+BL).The SHAM group and the OVX group received distilled water; and the OVX+E₂ group received 17-β- Estradiol 100μg/(kg·d),the OVX+BH group received BWS(1.2g/kg/day),the OVX+BL group received BWS(0.6g/kg/day)by the oral gavage daily for twelve weeks.After the last administration,the rats were used in following other experiments. The detection contents listed below:(1)Detective of the study and memory function by Morris water maze;(2)Measurement of serum estradiol;(3)Observation of the change of cellar morphology and ultrastructure in hippocampal subregion;(4) Analysis of CHAT,synaptophysin P38,Bcl-2,Bax positive cell expression by immunohistochemistry;(5)Analysis of CHAT,synaptophysin P38,GFAP,Arom mRNA expression by real-time PCR;(6)Analysis of GFAP,Arom protein expression by Western blot.Results1.Morris water maze:Spatial acquisition:In the spatial acquisition phase of testing, OVX group had significantly longer swim time and swim distant than SHAM group(P＜0.01).Compare to OVX group,E₂ group and BH group sigficantly reduced swim time and swim distant,which was close to swim time and swim distant of SHAM rats.BL group has a longer swim time and swim distant. Moreover,there was a significantly decreased trend for swim time and swim distant in SHAM,BH,BL group(P＜0.01),but no significantly decreased trend in OVX group(P＞0.05).There was no effect of block or treatment on swim speed, and no treatment by block interaction.In the spatial retention trial,both quadrant time and platform crossings were significantly affected by BWS and E₂.BWS and E₂ treatment protected against the OVX-induced decrease in the retention for the target quadrant during the trial,which was close to those of SHAM group.BH group had a decreased quadrant time and more platform crossing,compared to OVX group,and there was a statistical difference(P＜0.05).2.Comparision of serum E₂:The serum E₂ in SHAM group and E₂ group were significant higher than that in OVX group(P＜0.01).Treatment with BH and BL can improve the contents of serum E₂ statistically(P＜0.01).The level of serum E₂ in BH and BL groups is close to that of SHAM group(P＞0.05).3.Histological Analysis:Hippocamp tissue sections were stained with hematoxylin-eosin and the morphologic changes of the hippocampal pyramidal cells were observed under optical microscope.Each group appeared normal, showing no apparent neuronal damage.The cell cytoplasm appeared pink,the nucleus stained blue,both the celluar and nuclear membranes were well defined.4.Ultrastructural observation by TEM:Ultrastructural observation in the hippocampal CA1 region of the OVX group showed that the pyramidal cells were edematus severely,nucleus membranes crimpled or ruptured,chromatin aggregated, and the mitochondria swelled obviously,like empty-bubble,and the cristas broke and reduced or disappeared largely,whereas the mitochondria were protected well, and the changes were less prominent in BH,BL,E₂ groups like those of SHAM group.5.Spine synapse density measurement:Neuropil is a complex network structure composed by neuronic axons,dendrite,glial cells in cerebral cortex. Spine synapse density in neuropils was reduced in the OVX group relative to SHAM and E₂ groups(P＜0.01).Spine synapse density in BH group increased significantly compared to OVX group.BH group had no significant difference from the SHAM group(P＞0.05).The spine synapse density in BL group was higher than that in OVX group,but there was no statistical significance(P＞0.05).6.Immunohistochemical staining:Immunohistochemical staining demonstrated that positive cell of ChAT,synaptophysin P38,Bcl-2,Bax were all expressed in hippocampus CA1 region of rats. (1)Analysis result of image showed that density values of cholinergic neurons in SHAM group,E₂ group,BH group were higher statistically than that of OVX group(P＜0.01 or 0.05).The density value in BL group was higher than that in OVX group,but there was no significance in statistics(P＞0.05).The density values in BH group and BL group had no significant difference compared with SHAM group (P＞0.05).(2)Analysis result of image showed that density values of synaptophysin P38 increased significantly compared to that of OVX group(P＜0.01 or 0.05).The density value in BL group is higher than that of OVX group,but there was no statistical difference(P＞0.05).The density values in BH group,BL group and E₂ group had no significant difference compared with SHAM group(P＞0.05).(3)Analysis result of image showed that density value of Bcl-2 in OVX group decreased significantly compared to SHAM group(P＜0.01).Treatment with E₂,BH, BL can prevent this decline in OVX group significantly(P＜0.01 or 0.05).The density value in E₂,BH,BL group had no statistical difference compared to SHAM group(P＞0.05).(4)Analysis result of image showed that the density value of Bax in OVX group was higher significantly than that of SHAM group(P＜0.01).The density value in E₂,BH and BL group decreased significantly compare to SHAM group.There was no significant difference among E₂ group,BH group,BL group and SHAM group (P＞0.05).7.TUNEL staining:Apoptosis rates were detected with TUNEL.The apoptosis rate in OVX group increased significantly compare to SHAM group(P＜0.01).The apoptosis rates in E₂ group,BH group and BL group were lower statistically than that in OVX group(P＜0.01).There was no significant difference between E₂ group, BH group,BL group and SHAM group(P＞0.05).8.Expression of ChAT,synaptophysin P38,Bcl-2,Bax,Arom,GFAP mRNA in hippocampus CA1 region of rats were detected by real-time RT-PCR(1)Comparison of expression of ChAT mRNA:Compare to OVX group,the expression of ChAT mRNA in E₂ group and BH group increased significantly (P＜0.01),the expression of ChAT mRNA in BL group was higher than that in OVX group,but there was no significant difference between them(P＞0.05).The expression of ChAT in E₂ group and BH group had no statistical difference to that in SHAM group.(2)Comparison of expression of synaptophysin P38 mRNA:the expression of P38 mRNA in E₂ group and BH group increased significantly compared with OVX group (P＜0.01 or 0.05).The expression in BL group was higher than that in OVX group, but there was no statistical difference(P＞0.05).The expression in SHAM group had no significant difference compared with other groups except OVX group(P＞0.05).(3)Comparison of expression of Bcl-2 mRNA:the expression of Bcl-2 mRNA in OVX group was significantly lower than that in SHAM group(P＜0.01).The expression of Bcl-2 mRNA in E₂ group increased statistically compared to that in OVX group(P＜0.05).There was no significant difference between BH group and E₂ group(P＞0.05).The expression of Bcl-2 in BL group was higher than that in OVX group,but there was no statistical difference between them(P＞0.05).(4)Comparison of expression of Bax mRNA:the expression of Bax mRNA in OVX group was significantly higher than that in SHAM group(P＜0.01).The expression of Bax mRNA in E₂ group decreased statistically compared to that in OVX group (P＜0.05).There was no significant difference among BH group,BL group and E₂ group(P＞0.05).(5)Comparison of expression of Arom mRNA:The expression of Arom mRNA was higher significantly than that in SHAM group(P＜0.01).The expression of Arom mRNA in BH group decreased statistically compared to that in OVX group(P＜0.01). There was no statistical difference between SHAM group and BH group,E₂ group and BL group(P＞0.05).(6)Comparison of expression of GFAP mRNA:The expression of GFAP mRNA in OVX group increased statistically compared to SHAM group(P＜0.01).The expressions of GFAP mRNA in E₂ group,BH group,BL group were lower significantly than that in OVX group(P＜0.01).There was no significant difference between SHAM group and E₂ group,BH group,BL group(P＞0.05). 9.Comparison of expression of Arom and GFAP protein in hippocampus of rat(1)Comparison of expression of Arom protein:The expression of Arom protein in OVX group increased significantly compared to that in SHAM group(P＜0.01).The expression of protein in E₂ group,BH group,BL group decreased statistically compared to OVX group,especially that in BH group.There was no significant difference between SHAM group and BH group,BL group,E₂ group(P＞0.05).(2)Comparison of expression of GFAP protein:The expression of GFAP protein in OVX group increased statistically compared to that in SHAM group(P＜0.01).The expression of protein in E₂ group,BH group,BL group decreased significantly compared to OVX group,especially that in BH group.There was no statistical difference between SHAM group and BH group,BL group,E₂ group(P＞0.05).Conclusion1.BWS can ameliorate cognitional function of OVX rat.The mechanism may be associated with increasing the level of serum estradiol and protecting the mitochondrial function.2.Estrogen deficiency leads to descending expression of ChAT in cholinergic neuron, which may be one of the reasons of memory impairment of OVX rat.BWS can conduce cholinergic neuron to grow and survive.3.BWS can increase synaptic density in hippocampus of OVX rat and increase the expression of synaptophysin P38.The consistent result fully reflects the improvement of synaptic plasticity.This may be another reason of cognition improvement for BWS.4.One of the reasons for behavior abnormality in OVX rat may be descending expression of apoptosis inhibitor Bcl-2 and increasing expression of apoptosis promoter Bax.BWS can reverse ratio of Bcl-2/Bax,which leads cell to survive.5.The mechanism of improving memory for BWS may be associated with remaining stable of astrocytes and reducing the expression of Arom,then decreasing inflammatory response.